Therapeutic effects of mitoquinol during an acute heat stress challenge in growing barrows.

Study objectives were to determine the effects of mitoquinol (MitoQ, a mitochondrial-targeted antioxidant) on biomarkers of metabolism and inflammation during acute heat stress (HS). Crossbred barrows [n=32; 59.0±5.6 kg body weight (BW)] were blocked by BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n=8; TNCon), 2) TN and MitoQ (n=8; TNMitoQ), 3) HS control (n=8; HSCon), or 4) HS and MitoQ (n=8; HSMitoQ). Pigs were acclimated for 6 d to individual pens before study initiation. The trial consisted of two experimental periods (P). During P1 (2 d), pigs were fed ad libitum and housed in TN conditions (20.6±0.8°C). During P2 (24 h), HSCon and HSMitoQ pigs were exposed to continuous HS (35.2±0.2°C), while TNCon and TNMitoQ remained in TN conditions. MitoQ (40 mg/d) was orally administered twice daily (0700 and 1800 h) during P1 and P2. Pigs exposed to HS had increased rectal temperature, skin temperature, and respiration rate (+1.5°C, +6.8°C, and +101 breaths/min, respectively; P<0.01) compared to their TN counterparts. Acute HS markedly decreased feed intake (FI; 67%; P<0.01); however, FI tended to be increased in HSMitoQ relative to HSCon pigs (1.5 vs. 0.9 kg, respectively; P=0.08). Heat-stressed pigs lost BW compared to their TN counterparts (-4.7 vs. +1.6 kg, respectively; P<0.01); however, the reduction in BW was attenuated in HSMitoQ compared to HSCon pigs (-3.9 vs. -5.5 kg, respectively; P<0.01). Total gastrointestinal tract weight (empty tissue and luminal contents) was decreased in HS pigs relative to their TN counterparts (6.2 vs. 8.6 kg, respectively; P<0.01). Blood glucose increased in HSMitoQ relative to HSCon pigs (15%; P=0.04). Circulating non-esterified fatty acids (NEFA) increased in HS compared to TN pigs (P<0.01), although this difference was disproportionately influenced by elevated NEFA in HSCon relative to HSMitoQ pigs (251 vs. 142 µEq/L; P<0.01). Heat-stressed pigs had decreased circulating insulin relative to their TN counterparts (47%; P=0.04); however, the insulin:feed intake ratio tended to increase in HS relative to TN pigs (P=0.09). Overall, circulating leukocytes were similar across treatments (P>0.10). Plasma C-reactive protein remained similar among treatments; however, haptoglobin increased in HS relative to TN pigs (48%; P=0.03). In conclusion, acute HS exposure negatively altered animal performance, inflammation, and metabolism, which were partially ameliorated by MitoQ.

Calcium trafficking and gastrointestinal physiology following an acute lipopolysaccharide challenge in pigs.

The influence of systemic immune activation on whole-body calcium (Ca) trafficking and gastrointestinal tract (GIT) physiology is not clear. Thus, the study objectives were to characterize the effects of lipopolysaccharide (LPS) on Ca pools and GIT dynamics to increase understanding of immune-induced hypocalcemia, ileus, and stomach hemorrhaging. Twelve crossbred pigs [44 ±â€…3 kg body weight (BW)] were randomly assigned to 1 of 2 intramuscular treatments: (1) control (CON; 2 mL saline; n = 6) or (2) LPS (40 µg LPS/kg BW; n = 6). Pigs were housed in metabolism stalls to collect total urine and feces for 6 h after treatment administration, at which point they were euthanized, and various tissues, organs, fluids, and digesta were weighed, and analyzed for Ca content. Data were analyzed with the MIXED procedure in SAS 9.4. Rectal temperature and respiration rate increased in LPS relative to CON pigs (1.4 °C and 32%, respectively; P ≤ 0.05). Inflammatory biomarkers such as circulating alkaline phosphatase, aspartate aminotransferase, and total bilirubin increased in LPS compared with CON pigs whereas albumin decreased (P ≤ 0.02). Plasma glucose and urea nitrogen decreased and increased, respectively, after LPS (43% and 80%, respectively; P < 0.01). Pigs administered LPS had reduced circulating ionized calcium (iCa) compared to CON (15%; P < 0.01). Considering estimations of total blood volume, LPS caused an iCa deficit of 23 mg relative to CON (P < 0.01). Adipose tissue and urine from LPS pigs had reduced Ca compared to CON (39% and 77%, respectively; P ≤ 0.05). There did not appear to be increased Ca efflux into GIT contents and no detectable increases in other organ or tissue Ca concentrations were identified. Thus, while LPS caused hypocalcemia, we were unable to determine where circulating Ca was trafficked. LPS administration markedly altered GIT dynamics including stomach hemorrhaging, diarrhea (increased fecal output and moisture), and reduced small intestine and fecal pH (P ≤ 0.06). Taken together, changes in GIT physiology suggested dyshomeostasis and alimentary pathology. Future research is required to fully elucidate the etiology of immune activation-induced hypocalcemia and GIT pathophysiology.

Lipopolysaccharide (LPS) activates the immune system and this is accompanied with hypocalcemia and altered gastrointestinal tract (GIT) physiology. The study objectives were to characterize whole-body calcium (Ca) trafficking and evaluate GIT dynamics during LPS-induced immune activation. Ca concentrations were analyzed after intramuscular LPS injection. Administering LPS caused marked alterations in metabolic and inflammatory biomarkers and GIT dynamics, characterized by increased lower GIT motility and stomach hemorrhaging. Circulating Ca and adipose tissue and urine Ca output were decreased after LPS. Ca concentrations in other tissues and GIT contents were not detectably different. Thus, we were unable to account for about 110 mg Ca following LPS. Where and how circulating Ca is partitioned during immune activation remains unclear.

Investigating intestinal mast cell dynamics during acute heat stress in growing pigs.

Objectives were to examine the temporal pattern of intestinal mast cell dynamics and the effects of a mast cell stabilizer (ketotifen [Ket]) during acute heat stress (HS) in growing pigs. Crossbred barrows (n = 42; 32.3 ±â€…1.9 kg body weight [BW]) were randomly assigned to 1 of 7 environmental-therapeutic treatments: (1) thermoneutral (TN) control (TNCon; n = 6), (2) 2 h HS control (2 h HSCon; n = 6), (3) 2 h HS + Ket (2 h HSKet; n = 6); (4) 6 h HSCon (n = 6), (5) 6 h HSKet (n = 6), (6) 12 h HSCon (n = 6), or (7) 12 h HSKet (n = 6). Following 5 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (3 d), pigs were housed in TN conditions (21.5 ±â€…0.8 °C) for the collection of baseline measurements. During P2, TNCon pigs remained in TN conditions for 12 h, while HS pigs were exposed to constant HS (38.1 ±â€…0.2 °C) for either 2, 6, or 12 h. Pigs were euthanized at the end of P2, and blood and tissue samples were collected. Regardless of time or therapeutic treatment, pigs exposed to HS had increased rectal temperature, skin temperature, and respiration rate compared to their TNCon counterparts (1.9 °C, 6.9° C, and 119 breaths/min; P < 0.01). As expected, feed intake and BW gain markedly decreased in HS pigs relative to their TNCon counterparts (P < 0.01). Irrespective of therapeutic treatment, circulating corticotropin-releasing factor decreased from 2 to 12 h of HS relative to TNCon pigs (P < 0.01). Blood cortisol increased at 2 h of HS (2-fold; P = 0.04) and returned to baseline by 6 h. Plasma histamine (a proxy of mast cell activation) remained similar across thermal treatments and was not affected by Ket administration (P > 0.54). Independent of Ket or time, HS increased mast cell numbers in the jejunum (94%; P < 0.01); however, no effects of HS on mast cell numbers were detected in the ileum or colon. Jejunum and ileum myeloperoxidase area remained similar among treatments (P > 0.58) but it tended to increase (12%; P = 0.08) in the colon in HSCon relative to TNCon pigs. Circulating lymphocytes and basophils decreased in HSKet relative to TN and HSCon pigs (P ≤ 0.06). Blood monocytes and eosinophils were reduced in HS pigs relative to their TNCon counterparts (P < 0.01). In summary, HS increased jejunum mast cell numbers and altered leukocyte dynamics and proinflammatory biomarkers. However, Ket administration had no effects on mast cell dynamics measured herein.

Heat stress (HS) affects various physiological, metabolic, and endocrine parameters, ostensibly due to reduced intestinal barrier integrity and the ensuing immune response. Evidence indicates that generalized "stress" may be a critical component of HS-induced leaky gut, a mechanism likely mediated by mast cells. Mast cell activation has been extensively associated with various stress-related intestinal inflammatory conditions; however, its contribution to intestinal barrier dysfunction during HS remains unclear. Thus, this study was designed to evaluate mast cell dynamics during an acute HS challenge and to assess the effects a mast cell stabilizer on biomarkers of intestinal inflammation. Herein, HS induced a rapid increase in circulating cortisol, increased jejunum mast cell numbers, and altered metabolism, leukocyte dynamics, and proinflammatory biomarkers. Contrary to our hypothesis, HS did not alter circulating histamine (a biomarker of mast cell activation), and mast cell stabilization did not affect mast cell numbers nor altered histamine concentrations. Altogether, our observations support a connection between HS and intestinal mast cell infiltration that may contribute to the pathophysiology of intestinal dysfunction during a heat load.

Evaluation of the molecular response of corpora lutea to manganese-amino acid complex supplementation in gilts.

Porcine pregnancy establishment and maintenance are dependent on the formation of functional corpora lutea (CL). Manganese (Mn) is critical for CL function as it is a cofactor for Mn superoxide dismutase and enzymes involved in cholesterol synthesis. Previously, we have shown that luteal Mn content increased and luteal progesterone (P4) concentration decreased in the CL of gilts fed diets supplemented with an Mn-amino acid complex (Availa-Mn; Zinpro Corporation) compared with controls fed Mn sulfate. Importantly, serum P4 increased from 0 (estrus onset) to 12 d post estrus (dpe), as expected, but P4 abundance in circulation was not affected by dietary Mn source (P = 0.15). We hypothesized that a more bioavailable Mn source (which results in increased luteal Mn content) would alter the luteal proteome and abundance of mRNA associated with steroid biogenesis during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of the four gestation diets. The control diet (CON) contained 20 ppm of supplemental Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2), or 60 (TRT3) ppm of supplemental Mn in the form of a Mn-amino acid complex instead of Mn sulfate. Dietary treatment began at estrus synchronization (approximately 20 d before estrus) and continued through 12 dpe when gilts were euthanized and tissues were collected. Protein and total RNA extracts from the CL were used for proteomic analysis via label-free liquid chromatography with tandem mass spectrometry to assess global protein abundance and quantitative real-time polymerase chain reaction (qRT-PCR) to assess specific mRNA abundance, respectively. Compared with CON, 188, 382, and 401 proteins were differentially abundant (P < 0.10) in TRT1, TRT2, and TRT3, respectively. Gene Ontology enrichment software revealed that proteins involved in P4 signaling and cholesterol synthesis were downregulated in CL of gilts fed Mn-amino acid complex compared with controls. Quantitative RT-PCR showed that relative transcript abundance of genes encoding steroidogenic enzymes (CYP11A1 and StAR) in CL tissue was decreased in gilts from TRT2 compared with CON (P = 0.02), but TRT1 and TRT3 were not affected (P ≥ 0.30). Collectively, these data support our hypothesis that a more bioavailable dietary Mn source may influence luteal function by altering the abundance of protein and mRNA involved in steroidogenesis.

Impact of manganese amino acid complex on tissue-specific trace mineral distribution and corpus luteum function in gilts.

Functional corpora lutea (CL) are required for pregnancy establishment and gestational maintenance in swine, and CL function is susceptible to environmental influences. Manganese (Mn) could be critical in regulating CL function since it is a component of the antioxidant enzyme Mn superoxide dismutase (MnSOD) as well as enzymes involved in cholesterol and steroid hormone synthesis. We hypothesized that a more bioavailable dietary Mn source would increase Mn content in the CL thereby influencing luteal function during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of four gestation diets. The control diet (CON) met or exceeded National Research Council (2012) requirements and was formulated to contain 20 parts per million (ppm) of added Mn in the form of Mn sulfate. Three additional diets included 20 (treatment [TRT]1), 40 (TRT2), or 60 (TRT3) ppm of added Mn from a Mn-amino acid complex (Availa-Mn; Zinpro Corporation) instead of Mn sulfate. Dietary treatment began at estrus synchronization onset and continued through 12 days post estrus (dpe) of the ensuing estrous cycle. Blood samples were collected at estrus onset, which was assigned as 0 dpe, as well as 4, 8, and 12 dpe. Gilts were euthanized and tissues were collected at 12 dpe. Serum progesterone (P4) increased (P < 0.01) from 0 to 12 dpe but was unaffected by dietary treatment (P = 0.15) and there was no effect of the interaction between day and treatment (P = 0.85). Luteal Mn content increased (P ≤ 0.05) by 19%, 21%, and 24% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Luteal P4 concentrations decreased (P = 0.03) 25%, 26%, and 32% in gilts fed TRT1, TRT2, and TRT3, respectively, compared to CON. Relative to CON gilts, CL calcium content decreased (P = 0.02) by 36%, 24%, and 34% for TRT1, TRT2, and TRT3 gilts, respectively. Collectively, these data support the hypothesis that feeding a more bioavailable Mn source increases Mn accumulation in CL tissue. If and how this influences CL function may be related to altered luteal P4 concentrations.

Effects of continuously infusing glucose or casein into the terminal ileum on biomarkers of metabolism, inflammation, and intestinal morphology in growing pigs.

Study objectives were to determine the effects of continuously infusing glucose (GLC) or casein (CAS) into the terminal ileum on biomarkers of metabolism, inflammation, and intestinal morphology in growing pigs. Crossbred gilts (n = 19; 81 ± 3 kg body weight [BW]) previously fitted with T-cannulas at terminal ileum were used in the current experiment. Following 4 d of acclimation, pigs were enrolled in 2 experimental 4-d periods (P). During P1, pigs were housed in individual pens and fed ad libitum for collection of baseline parameters. At the beginning of P2, pigs were assigned to 1 of 3 infusion treatments: 1) control (CON; water; 3 liters/d; n = 7), 2) GLC (dextrose 50%; 500 g/d; n = 6;), or 3) CAS (casein sodium salt; 300 g/d; n = 6). Water, GLC, and CAS solutions were continuously infused at a rate of 125 mL/h for the entirety of P2. Animals were euthanized at the end of P2, and intestinal tissue was collected. During P2, average daily feed intake differed across treatments and was reduced in GLC compared with CON pigs (14%), while CAS pigs consumed an intermediate amount (P = 0.05). Average daily gain and final BW were similar across treatments. A treatment by time interaction was observed for blood urea nitrogen (BUN; P < 0.01), as it decreased in GLC (21%) while it gradually increased in CAS (76%) pigs relative to CON pigs. Mild hyperthermia occurred with both GLC and CAS infusions relative to CON (+0.3 and 0.2 °C, respectively; P < 0.01). Blood neutrophils increased in CAS relative to CON pigs (26%) but remained similar between CON and GLC treatments (P < 0.01). Blood monocytes decreased in GLC relative to CON pigs (24%) while CAS pigs had an intermediate value (P = 0.03). Circulating lipopolysaccharide binding protein tended to decrease in GLC (29%) relative to CON pigs but remained similar between CON and CAS pigs (P = 0.10). Plasma tumor necrosis factor-alpha was similar across treatments. Ileum villus height:crypt depth was increased in CAS compared with CON pigs (33%; P = 0.05) while GLC pigs had an intermediate value. Colon myeloperoxidase-stained area increased in CAS compared with CON pigs (45%; P = 0.03) but remained similar between GLC and CON pigs. In summary, continuously infusing GLC or CAS into the terminal ileum appeared to stimulate a mild immune response and differently altered BUN patterns but had little or no effects on blood inflammatory markers, intestinal morphology, or key production parameters.

Rapamycin administration during an acute heat stress challenge in growing pigs.

Study objectives were to determine the effects of rapamycin (Rapa) on biomarkers of metabolism and inflammation during acute heat stress (HS) in growing pigs. Crossbred barrows (n = 32; 63.5 ± 7.2 kg body weight [BW]) were blocked by initial BW and randomly assigned to 1 of 4 environmental-therapeutic treatments: 1) thermoneutral (TN) control (n = 8; TNCon), 2) TN and Rapa (n = 8; TNRapa), 3) HS control (n = 8; HSCon), or 4) HS and Rapa (n = 8; HSRapa). Following 6 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (10 d), pigs were fed ad libitum and housed in TN conditions (21.3 ± 0.2°C). During P2 (24 h), HSCon and HSRapa pigs were exposed to constant HS (35.5 ± 0.4°C), while TNCon and TNRapa pigs remained in TN conditions. Rapamycin (0.15 mg/kg BW) was orally administered twice daily (0700 and 1800 hours) during both P1 and P2. HS increased rectal temperature and respiration rate compared to TN treatments (1.3°C and 87 breaths/min, respectively; P < 0.01). Feed intake (FI) markedly decreased in HS relative to TN treatments (64%; P < 0.01). Additionally, pigs exposed to HS lost BW (4 kg; P < 0.01), while TN pigs gained BW (0.7 kg; P < 0.01). Despite marked changes in phenotypic parameters caused by HS, circulating glucose and blood urea nitrogen did not differ among treatments (P > 0.10). However, the insulin:FI increased in HS relative to TN treatments (P = 0.04). Plasma nonesterified fatty acids (NEFA) increased in HS relative to TN treatments; although this difference was driven by increased NEFA in HSCon compared to TN and HSRapa pigs (P < 0.01). Overall, circulating white blood cells, lymphocytes, and monocytes decreased in HS compared to TN pigs (19%, 23%, and 33%, respectively; P ≤ 0.05). However, circulating neutrophils were similar across treatments (P > 0.31). The neutrophil-to-lymphocyte ratio (NLR) was increased in HS relative to TN pigs (P = 0.02); however, a tendency for reduced NLR was observed in HSRapa compared to HSCon pigs (21%; P = 0.06). Plasma C-reactive protein tended to differ across treatments (P = 0.06) and was increased in HSRapa relative to HSCon pigs (46%; P = 0.03). Circulating haptoglobin was similar between groups. In summary, pigs exposed to HS had altered phenotypic, metabolic, and leukocyte responses; however, Rapa administration had limited impact on outcomes measured herein.

Effects of an oral supplement containing calcium and live yeast on post-absorptive metabolism, inflammation and production following intravenous lipopolysaccharide infusion in dairy cows.

Objectives were to evaluate the effects of an oral supplement containing soluble Ca, and live yeast in LPS-challenged dairy cows. The trial consisted of 2 experimental periods (P). During P1 (3 d), cows (n = 12) were fed ad libitum and baseline data was collected. At the beginning of P2 (which lasted 96 h), all cows were i.v. challenged with 0.375 µg/kg BW LPS. Cows were assigned randomly to 1 of 2 treatments: 1) control (CON; no bolus; n = 6) or 2) an oral bolus containing Ca and live yeast (CLY; YMCP Vitall® 44.718 g of elemental Ca; TechMix, LLC., Stewart, MN; n = 6), administered -0.5 and 6.5 h relative to LPS infusion. Following LPS administration, circulating Ca decreased in both treatments but supplemental CLY ameliorated the hypocalcemia (48 h area under the curve: -10.8 vs. -1.9 mmol/L × h; P < .01). Lipopolysaccharide decreased dry matter intake (DMI; 60%) similarly for both treatments on d 1, but overall (d 1-4) DMI tended to be reduced less (14 vs. 30%; P = .06) in CLY supplemented vs CON cows. Lipopolysaccharide reduced milk yield (70%; P < .01) from 12 to 24 h, but throughout P2, milk yield from CLY supplemented cows was increased (38%; P = .03) relative to CON cows. Overall during P2, circulating LPS-binding protein and serum amyloid A increased post LPS (3- and 4-fold, respectively, P < .01), but were unaffected by treatment (P ≥ .68). In conclusion, providing an oral supplement containing Ca and live yeast prior to and following LPS administration markedly ameliorated LPS-induced hypocalcemia and improved DMI and milk yield.